update on animal models of diabetic retinopathy: from ...increased flux through the polyol pathway,...

PERSPECTIVE

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IntroductionDiabetic retinopathy (DR), a major complication of diabetesmellitus, is one of the leading causes of blindness worldwide. Earlydiagnosis and prevention of retinopathy in diabetic individuals iscrucial for preventing vision loss. Prolonged hyperglycemia causesirreversible pathological changes in the retina, leading toproliferative DR with retinal neovascularization and diabeticmacular edema (DME) in some individuals (Mohamed et al., 2007;Cheung et al., 2010).

Treatment of DR can only be achieved through an enhancedunderstanding of disease pathogenesis; however, because moststructural, functional and biochemical studies cannot be carriedout in human subjects, animal models are essential for studyingDR pathology, and thus for developing new and better treatments.

Clinical features of DRDR is widely regarded as a microvascular complication of diabetes.Clinically, DR can be classified into non-proliferative DR (NPDR)and proliferative DR (PDR) (Cheung et al., 2010). NPDR ischaracterized ophthalmoscopically by the presence ofmicroaneurysms and dot and blot hemorrhages (Fig. 1A). NPDRhas been further subdivided into progressive stages: mild, moderateand severe. Severe NPDR (also called preproliferative DR) showsincreased retinal microvascular damage as evidenced by cottonwool spots, venous beading, venous loops and intra-retinalmicrovascular abnormalities (IRMAs). Capillary non-perfusion anddegeneration of the retina can be detected in individuals withdiabetes following intravascular injection of fluorescein. If leftuntreated, PDR (characterized by abnormal retinalneovascularization) can develop (Fig. 1B). A clinically importantoutcome of PDR is retinal and vitreous hemorrhage and tractionalretinal detachment (Cheung et al., 2010).

DME can occur at any stage (i.e. along with NPDR or PDR) andis now the most common cause of vision loss due to DR (Cheunget al., 2010).

Epidemiology and risk factorsDiabetes affects more than 300 million people worldwide, and isexpected to affect an estimated 500 million by 2030 (InternationalDiabetes Federation, 2011). Studies have shown that nearly allindividuals with type 1 diabetes [also known as insulin-dependentdiabetes mellitus (IDDM)] and more than 60% of individuals withtype 2 diabetes (non-insulin-dependent diabetes mellitus) havesome degree of retinopathy after 20 years. Current population-

Disease Models & Mechanisms 5, 444-456 (2012) doi:10.1242/dmm.009597

Update on animal models of diabetic retinopathy:from molecular approaches to mice and highermammalsRemya Robinson1,*, Veluchamy A. Barathi1,2,*, Shyam S. Chaurasia1, Tien Y. Wong1,2,3,‡ and Timothy S. Kern4,5

1Singapore Eye Research Institute, and 3Singapore National Eye Centre, 11 ThirdHospital Avenue, Singapore 168751, Singapore2Department of Ophthalmology, Yong Loo Lin School of Medicine, NationalUniversity of Singapore, Singapore 119074, Singapore4School of Medicine, Case Western Reserve University, Cleveland, OH 44106, USA5Stokes Veterans Administration Hospital, Cleveland, OH 44106, USA*These authors contributed equally to this work‡Author for correspondence ([email protected])© 2012. Published by The Company of Biologists LtdThis is an Open Access article distributed under the terms of the Creative CommonsAttribution Non-Commercial Share Alike License (http://creativecommons.org/licenses/by-nc-sa/3.0), which permits unrestricted non-commercial use, distribution and reproductionin any medium provided that the original work is properly cited and all furtherdistributions of the work or adaptation are subject to the same Creative Commons Licenseterms.

Diabetic retinopathy (DR) is the most common microvascular complication of diabetes and one of the major causes ofblindness worldwide. The pathogenesis of DR has been investigated using several animal models of diabetes. Thesemodels have been generated by pharmacological induction, feeding a galactose diet, and spontaneously by selectiveinbreeding or genetic modification. Among the available animal models, rodents have been studied most extensivelyowing to their short generation time and the inherited hyperglycemia and/or obesity that affect certain strains. Inparticular, mice have proven useful for studying DR and evaluating novel therapies because of their amenability togenetic manipulation. Mouse models suitable for replicating the early, non-proliferative stages of the retinopathy havebeen characterized, but no animal model has yet been found to demonstrate all of the vascular and neuralcomplications that are associated with the advanced, proliferative stages of DR that occur in humans. In this review, wesummarize commonly used animal models of DR, and briefly outline the in vivo imaging techniques used forcharacterization of DR in these models. Through highlighting the ocular pathological findings, clinical implications,advantages and disadvantages of these models, we provide essential information for planning experimental studies ofDR that will lead to new strategies for its prevention and treatment.

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Disease Models & Mechanisms 445

Animal models of diabetic retinopathy PERSPECTIVE

based studies suggest that about one-third of the diabeticpopulation have signs of DR and approximately one tenth havevision-threatening stages of retinopathy, including PDR and DME(Wong et al., 2006; Wong et al., 2008; Wang et al., 2009; Zhang etal., 2010).

People with diabetes are 25 times more likely to become blindthan non-diabetics. In fact, reports have shown that 50% ofdiabetics will become blind within 5 years following diagnosis ofPDR, if left untreated (Ciulla, 2004; Klein, 2008; Wong et al., 2009).The number of people with DR is rapidly increasing owing to adramatic rise in the prevalence of type 2 diabetes, reflecting theincreased prevalence of obesity and metabolic syndrome observedin recent years (Cheung et al., 2010; Raman et al., 2010).

The three major risk factors for DR are prolonged (1) diabetes,(2) hyperglycemia and (3) hypertension, which have been shownto be consistently associated with DR in epidemiological studiesand clinical trials (Wong et al., 2006; Wong et al., 2008; Wang etal., 2009; Cheung et al., 2010; Grosso et al., 2011). Dyslipidemiaand body mass index might also be risk factors for DR, butassociations have not been as consistent (Lim and Wong, 2011;Benarous et al., 2011; Dirani et al., 2011; Sasongko et al., 2011).Emerging evidence supports a genetic component for DR, butspecific genes associated with the disease have not been clearlyidentified despite large studies (Liew et al., 2006; Abhary et al., 2009;Sobrin et al., 2011). It remains difficult to predict which diabeticindividuals will progress from NPDR to PDR.

Pathophysiology of DRThe pathogenesis of the development of DR is highly complexowing to the involvement of multiple interlinked mechanismsleading to cellular damage and adaptive changes in the retina (Frank,2004). Hence, the fundamental cause(s) of DR has not beenelucidated completely despite years of clinical and laboratoryinvestigation. In the past, retinopathy has been characterizedlargely by its microvascular abnormalities, including endothelial celldysfunction, vessel leakage, and vascular occlusion anddegeneration (Curtis et al., 2009). Recent evidence, however,indicates that retinal complications of diabetes are a composite offunctional and structural alterations in both the microvascular andneuroglial compartments (Antonetti et al., 2006; Curtis et al., 2009;Villarroel et al., 2010; Barber et al., 2011). The exact mechanismsby which hyperglycemia initiates the vascular or neuronalalterations in retinopathy have not been completely defined (Curtiset al., 2009; Villarroel et al., 2010). The cellular damage in the retinahas been speculated to be caused by several mechanisms, including

increased flux through the polyol pathway, production of advancedglycation end products (AGEs), increased oxidative stress andactivation of the protein kinase C (PKC) pathway, but many of thesehypotheses have yet to be validated in human studies or clinicaltrials (Frank, 2004; Cheung et al., 2010).

DR also shares similarities with chronic inflammatory diseases:it causes increased vascular permeability, edema, inflammatory cellinfiltration, tissue destruction, neovascularization, and theexpression of pro-inflammatory cytokines and chemokines in theretina. Increased expression of vasoactive factors and cytokinesprobably plays an important role in mediating the structural andfunctional changes in the retina (Khan and Chakrabarti, 2007;Wirostko et al., 2008). Recent studies strongly suggest thatinflammation is also important in the pathogenesis of early stagesof experimental DR (Kern, 2007; Liou, 2010; Tang and Kern, 2011),although studies in humans have not found a consistent associationbetween systemic markers of inflammation and retinopathy(Nguyen et al., 2009; Lim et al., 2010). Thus, it remains uncertainwhether inflammation also plays a crucial role in the developmentand progression of DR in humans. Some of the major pathwaysand factors involved in the pathogenesis of DR are shown in Fig. 2.

Outstanding questions regarding DR etiology andtreatment The currently available treatments for advanced stages of DR,including PDR and DME, are laser photocoagulation, surgicalvitrectomy or intraocular injections of steroids and vascularendothelial growth factor (VEGF) inhibitors. Although thesetreatments have had high success rates, they are not useful for earlystages of DR, and do not completely eliminate the risk of blindness(Cheung et al., 2010). Laser therapy is inherently destructive, withunavoidable side effects, and is not effective in reversing vision loss.The new approaches involving anti-VEGF therapy also havepotential ocular and systemic risks (Cheung et al., 2010; Truong etal., 2011). Thus, new treatment strategies that are preventativeand/or can provide interventions earlier in diabetes to delay orprevent the progression of early NPDR are needed.

In this regard, it is crucial to more fully establish the underlyingmechanisms and causes for DR. Studies suggest that multifactorialapproaches intervening at the hemodynamic, metabolic andcytokine levels will delay the development of DR. However, the basicfundamental mechanism(s) of hyperglycemic influence orregulation of retinal vessels is not completely understood. Despitethe large number of experimental studies being conducted on theetiology and treatment of DR in various laboratories, some of theimportant issues that remain unanswered are: (1) the mechanismsthat link neural impairment in early diabetes to the developmentof retinal vascular abnormalities; (2) the potential role ofinflammation in diabetes-induced retinal neurodegeneration; (3)the specific retinal layers that are vulnerable to metabolic imbalancein DR; and (4) the genetic basis of DR.

Further studies using appropriate animal models are requiredto provide answers to some of these questions, which will providethe basis for new treatment approaches that prevent or delay theonset of DR.

Fig. 1. Clinical features of DR. Fundus photographs of human patientsshowing (A) early non-proliferative diabetic retinopathy (NPDR) and (B)proliferative diabetic retinopathy (PDR).

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Animal models of diabetic retinopathyPERSPECTIVE

Animal models of DRDuring the last two decades, extensive research with animal modelsof diabetes has been carried out. To date, several species – includingmice, rats, cats, dogs, pigs and non-human primates – have beenused as models to provide valuable information on the cellular andmolecular aspects of pathogenesis of DR. Diabetes in animals isusually induced with chemicals such as alloxan or streptozotocin(STZ), by surgical pancreatectomy, or spontaneously by selectivebreeding or genetic manipulation. Most experimental studies onDR to date have used animal models of type 1 diabetes (Cheta,1998; Kern, 2009; Zheng and Kern, 2010). Recently, many transgenicand knockout mouse models have also been developed to studythe molecular pathways involved in DR pathology. In the followingsections, we review currently available animal models of DR,beginning with a description of the various techniques used for invivo characterization. We then briefly discuss studies of DRperformed using each model, comment on their contributions toinvestigating the pathogenesis of DR, and highlight their advantagesand disadvantages. Retinal lesions of DR observed in small andhigher animal models are summarized in Tables 1 and 2,respectively.

Characterization of DR in animal modelsThe use of appropriate imaging techniques to analyze animalmodels of human DR is essential to experimentally study andcharacterize the disease. Eyes collected at necropsy can be subjectedto sensitive histological techniques (such as trypsin digest) to studythe retinal vasculature in detail. In addition, various molecular and

biochemical techniques (such as quantitative PCR, microarray,western blotting, as well as protein, enzyme and cytokine assays)can be carried out to study the expression of various genes andproteins in the eyes. While the animals are alive, the vascular andnon-vascular alterations of DR (Cunha-Vaz, 2007) can be monitoredin the same animal over time using various techniques, includingelectrophysiological testing, fundus photography (FP), fundusfluorescein angiography (FFA) and optical coherence tomography(OCT); these techniques are briefly discussed below.

Electrophysiological testsElectrophysiological tests of retinal function are the mostcommonly used non-invasive technique for studying visual functionin animal models. These tests include electroretinography (ERG),pattern ERG (PERG), multi-focal ERG (mfERG) and visually evokedpotential. Studies using ERG revealed functional abnormalitiesbefore the evidence of vascular changes in the eyes of diabetic rats(Kohzaki et al., 2008). Alterations in retinal electrical responses,which arise from the neural retina, are one of the earliestmanifestations of DR. ERG studies on diabetic animals have shownreduced a- and b-wave amplitude (Li et al., 2002), and prolongedimplicit time in the oscillatory potentials (OPs) (Hancock and Kraft,2004).

FP and FFARetinal vasculature photographs obtained with FP provide arelatively insensitive tool to monitor the progression of DR, butare adequate for providing important information about the severity

Activation of cytokines and growth factors

Dysfunction of vascular

and neuronal cells

Retinal hypoxia

Neovascularistion

Hyperglycemia

AGEs Oxidative stress PKC activation Polyol pathway Inflammation

Vascular

permeability

Neural

dysfunction

Mild NPDR

Pro

gre

ssio

n

Microaneurysms

Moderate NPDR

PDR

Intraretinal hemorrhages,

venous beading, basement

membrane thickening and

microvascular abnormalities

Neovascularization,

retinal detachment

Vision loss

Clin

ica

l sym

pto

ms

Severe NPDR

Macular edema

Neovascularization,

retinal detachment

Microaneurysms,

microvascular lesions,

capillary microangiopathy

Diabetic

retinopathy

Fig. 2. Flow diagram showing the major key factors involved in the pathogenesis of DR and the clinical symptoms evident at different stages of DR. DRis a multifactorial disease involving several pathological mechanisms, including increased oxidative stress, inflammation, the polyol pathway leading to sorbitolaccumulation, production of advanced glycation end products (AGEs) and activation of the protein kinase C (PKC) pathway. These pathways can in turn activatethe production of cytokines and many vasoactive factors, such as vascular endothelial growth factor (VEGF) and pigment epithelium-derived factor (PEDF),which are vital in mediating the structural and functional changes of DR. Clinically significant diabetic macular edema (DME) can occur in the late stages of DRwith non-proliferative or proliferative retinopathy and is the most common cause of vision loss.

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Table 1. Retinal lesions reported in small animal models of DR

Animal model Type of diabetes

Age of onset of

diabetes Retinal lesions References

Rats

STZ Type 13 days after STZ

injection

• Pericyte loss

• Vascular leakage

• Blood retinal barrier breakdown

• Ganglion cell loss

• Endothelial cell damage

• Vascular occlusion of retinal capillaries

• Thicker capillary BM

Robison et al., 1991; Anderson et al.,1995; Miyamoto et al., 1999; Xu et al.,

2004; Gastinger et al., 2006; Zhengand Kern, 2010

Galactosemia – –

• Pericyte loss

• Acellular capillaries

• IRMAs


Kern and Engerman, 1995

BB Type 1 2-5 months

• Pericyte loss


• Blood retinal barrier breakdown


Blair et al., 1984; Sima et al., 1985

WBN/Kob Type 1 9-21 months• Acellular capillaries

• Thicker capillary BMMiyamura et al., 1998; Bhutto et al.,

1999; Tsuji et al., 2009

SDT Type 1 5-6 months

• Pericyte loss



• Retinal detachment with fibrousproliferation

Yamada et al., 2005; Kakehashi et al.,2006; Sasase et al., 2010

ZDF Type 2 1-2 months

• Pericyte loss



Danis et al., 1993; Yang et al., 2000;Behl et al., 2008

OLETF Type 2 4-5 months

• Microaneurysms

• Tortuosity

• Loop formations of capillaries

• Vessel caliber irregularity


Bhutto et al., 2002; Lu et al., 2003

GK Type 2 1-2 months • Increased EC:pericyte ratios Agardh et al., 1997

Mice

STZ Type13 days after STZ

injection

• Pericyte loss


• Apoptosis of vascular cells


• Thinning of retina

Martin et al., 2004; Leichman et al.,2005

Galactosemia

• Pericyte loss


• Microaneurysms


Kern and Engerman, 1996a

NOD Type 1 8 months• Loss of retinal microvessels

• Disordered focal proliferation of newvessels

Makino et al., 1980; Shaw et al., 2006;Lee et al., 2008

db/db Type 2 1-2 months

• Pericyte loss


• Blood-retinal barrier breakdown


Midena et al., 1989; Clements et al.,1998; Cheung et al., 2005

Ins2Akita Type 1 1 month

• Increased vascular permeability


• Thinning of retina


Barber et al., 2005; Gastinger et al.,2008

Akimba Type 1 1 month

• Microaneurysms


• Venous beading

• Tortuosity

• Capillary dropout

• Hemorrhage

• Possible neovascularization

• Retinal edema

Rakoczy et al., 2010

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of retinopathy in patients, including vessel caliber changes, swellingof vessels, abnormal new growth of vessels on retinal surface andtortuosity. FFA, which monitors the flow of a fluorescent dyethrough the retinal vasculature, is useful for demonstrating vascularabnormalities, non-perfusion and vascular leakage. Neither FP norFFA has been extensively used in animal studies, owing to the rapiddevelopment of cataracts in many species, and the fact that smallanimals’ eyes are small, highly curved globes, which prevent lightrays from focusing on the retina. Moreover, FFA cannot be usedeasily in albino animals or in the non-pigmented portions of retinain animals having a tapetum (dogs, cats) (Hawes et al., 1999). Fig.3 shows a comparison of FFA performed in both B6 wild-type(pigmented) and Balb/c albino (non-pigmented) mice.

OCTMeasurement of retinal thickness has been used as an indicator ofprogressive neural retinal pathology in animal models of retinaldegeneration, but the degree of degeneration that occurs in diabeticanimals is considerably less than in retinal degeneration modelsand is more difficult to measure reproducibly. High-resolution OCTmeasures retinal thickness non-invasively in rodents (as in humans),and recent machines can produce images with some features ofretinal histology. Studies have also shown that ERG amplitude lossis highly correlated with retinal thinning as measured by OCT (Liet al., 2001; Fischer et al., 2009). Similarly, reduced retinal thicknesshas been demonstrated in diabetic mice using OCT (Rakoczy etal., 2010). Retinal thickening has been detected with OCT in

monkeys (Sakurai et al., 2009) and dogs (Panzan et al., 2004), and,using magnetic resonance imaging, also in diabetic rats (Berkowitzet al., 2007). Detailed validation of structural and functional lesionsof DR in mice is available in a review by Kern et al. (Kern et al.,2010a).

Emerging techniques to measure retinal vascular caliberHuman epidemiological studies have shown that measurement ofretinal vascular caliber from photographs might provide clues toearly pathological processes in pre-diabetes, diabetes and DR(Nguyen et al., 2007; Nguyen et al., 2008; Rogers et al., 2008; Wong,2011). An insight into the retinal vessel caliber changes in animalmodels of DR could thus potentially help to characterize thestructure and pathology of the microcirculation, and to examineits relationship to systemic vascular diseases in diabetes. Currently,there are no well-established methods to study the retinal vesselcaliber changes in animal models. Recently, a semi-automatedcomputer-based quantitative program to measure retinal vascularcaliber from retinal photographs of rodents has become available,which is identical to the program used in large epidemiologicalstudies of both diabetic and non-diabetic human populations (Fig.4A,B). This novel imaging software measures subtle retinal vesselcaliber changes in rodents (Fig. 4C,D), which might be markers ofearly microvascular dysfunction in diabetes. Such developmentswill open the door for advanced quantitative assessments in animalmodels, which could substantially contribute to a betterunderstanding of the pathogenesis and prediction of DR.

Table 2. Retinal lesions of DR reported in higher animal models of DR

Animal models Type of diabetes Retinal lesions References

Dog Type 1

• Pericyte loss

• Microaneurysms


Gardiner et al., 1994; Kern and Engerman, 1996b

Galactosemic dogs –

• Pericyte loss


• Microaneurysms

• IRMAs

• Retinal hemorrhages

Takahashi et al., 1993; Kador et al., 1995; Kern and Engerman 1996b; Kobayashi et al., 1998

Cat Type 1


• Microaneurysms


• Tortuosity


Mansour et al., 1990; Hatchell et al., 1995; Linsenmeier et al., 1998; Budzynski et al., 2005

Pig Type 1 • Retinal microvasculopathy

• Thicker capillary BM Hainsworth et al., 2002; Lee et al., 2010

Non-human primate

Type 1

• Microaneurysms

• IRMAs

• Cotton wool spots

• Macular edema

Tso et al., 1988

Type 2


• Cotton wool spots

• Intraretinal hemorrhages

• Microaneurysms

• IRMAs

• Hard exudates in the macula

• Decreased RGC layer

Kim et al., 2004; Johnson et al., 2005

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Rodent models of DRVarious rodent models have been used for studying the molecularmechanisms underlying the pathogenesis of DR. These models areeasy to handle, relatively inexpensive, have short reproductive cyclesand have a similar genetic background to humans, hopefullyallowing experimental results to be extrapolated. Rodent modelsof DR vary with respect to species (predominantly rat or mouse),strain, method of diabetic induction and duration of diabetes.Advanced techniques of genetic manipulation, such as tissue-specific transgenic expression and targeted gene knockout, haveincreased the relative importance of mouse models for experimentsthat specifically require genetically engineered models. Thus, theseanimals provide a remarkable platform to investigate thepathogenesis of at least the early stages of the retinopathy, becausegenetic alterations of selected metabolic and pathophysiologicalmechanisms are now possible.

However, a major criticism of using rodents to model DR is thatthey might not exactly mirror the human condition, especially withregard to the extent of pathology. Rodent models reproduce most

aspects of the early stages of DR, but have not been found toreproducibly develop the late, neovascular stage of the disease,probably owing to the short lifespan of the animals and thus theshorter duration of diabetes.

Chemically induced diabetic modelsDiabetes can be induced in animals using STZ or alloxan, whichdestroy pancreatic -cells and thereby induce type 1 diabetes.Rodents that have been made diabetic in this manner have beenstudied for up to 24 months in a hyperglycemic yet healthycondition, by providing them with small amounts of insulin everyfew days. This approach reproduces early symptoms of DR, suchas loss of retinal pericytes and capillaries, thickening of the vascularbasement membrane (BM), vascular occlusion and increasedvascular permeability (Kern and Mohr, 2007; Kern, 2009; Zhengand Kern, 2010). Physiological and biochemical changes in theretina begin to appear between 1-2 months after the onset ofhyperglycemia in STZ-induced diabetic rats. Diabetes-inducednon-vascular changes (neuronal and glial) are seen before thedevelopment of changes in vascular cells and might contribute tothe pathology of the vascular disease in this model (Barber et al.,1998; Zeng et al., 2001; Kohzaki et al., 2008). However, there arevariations in the reported retinal biochemistry andhistopathological response to diabetes between species and alsowithin the same species. In fact, a recent report studied thedifferences in the rate at which early stages of DR develop in threedifferent rat strains (Sprague Dawley, Lewis and Wistar) withdiabetes induced by STZ. After 8 months of diabetes, Lewis ratsshowed the most accelerated loss of retinal capillaries and retinalganglion cells (RGCs), whereas Wistar rats showed degenerationof the capillaries without significant neurodegeneration andSprague Dawley rats showed no lesions at this time point (Kern etal., 2010b).

STZ-induced mouse models were not frequently used for studieson DR in the past because it was more difficult to induce diabetesin mice than in rats and was difficult to keep the tiny animals aliveonce diabetic. These problems have been overcome more recently.STZ-induced diabetic B6 mice demonstrated acellular capillaries,apoptosis of vascular cells and pericyte ghosts in the retina, thehallmark of early characteristics of DR (Feit-Leichman et al., 2005),at ~6 months after the onset of diabetes. Advanced proliferativeretinal changes did not develop in these mice within the studyduration (18 months of diabetes). Whether or not mice developloss of RGCs is controversial. At 10-14 weeks after STZ treatment,these mice demonstrated loss of RGCs, and significant thinning ofthe inner and outer layers of the retina (Martin et al., 2004; Barberet al., 2005). Other studies, however, found no evidence of RGCloss in diabetic mice (Asnaghi et al., 2003; Feit-Leichman et al., 2005;Gastinger et al., 2006). These diabetic models are mostly used todemonstrate early changes of DR. Studies of advanced proliferativeretinal changes cannot be carried out in these models because theydie before PDR could be detected.

eNOS–/– miceRecently, the effects of single genes on the development of DR havebeen assessed by inducing diabetes with STZ in transgenic or geneknockout mice. For example, Li et al. investigated the pathogenicrole of endothelial nitric oxide synthase (eNOS) dysfunction in the

Fig. 3. Fundus fluorescein angiography (FFA) images show thecomparison between normal B6 (pigmented) and Swiss albino (non-pigmented) mice. Normal (A) B6 (pigmented) and (B) Swiss albino (non-pigmented) mice. FFA cannot be used in albino mice owing to the absence ofpigment, which produces severe glare.

Fig. 4. Retinal vascular caliber measurement. The measurement of retinalvessel caliber in human (A,B) and mouse (C,D) with fundus imaging, usingsemi-automated computer-based quantitative program (SIVA). The whitearrow indicates the artery, the black arrow indicates the vein and an asterisk (*)shows the optic nerve head.

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development of DR by inducing diabetes using STZ in eNOSknockout (eNOS–/–) mice (Li et al., 2010). The retinal vasculatureof eNOS–/– mice develops normally and is associated with increasedvascular-associated neuronal NOS activity that compensates forthe eNOS deficiency in the retina. STZ-induced diabetes in thesemice showed accelerated retinal complications of DR whencompared with age-matched STZ-induced diabetic B6 wild-typemice. The retinal complications included increased vessel leakage,gliosis, acellular retinal capillaries and retinal capillary BMthickening. Further studies using this model will be useful forinvestigating the cellular and molecular mechanisms of DR,including gliotic responses in retinal Muller cells.

Spontaneously diabetic rat modelsZucker diabetic fatty ratsZucker diabetic fatty (ZDF) rats are genetic models of type 2 (non-insulin-dependent) diabetes and become hyperglycemic at 6-7weeks of age. These rats usually die at ~1 year of age, but can bemaintained without treatment if supplemented with glucose ofmore than 500 mg/dl. Studies demonstrated pericyte loss, a thickerretinal capillary BM, and an increased number of endothelialintercellular junctions and focal nodules in ZDF rats. This modelis thought to be useful for pharmacological intervention studiesbecause it is naturally and severely type 2 diabetic, showingquantifiable retinal vascular changes. In addition, same-sexlittermates can be used as controls (Danis and Yang, 1993; Ottleczet al., 1993; Yang et al., 2000).

WBN/Kob ratsThe WBN/Kob rat is also a spontaneously type 2 diabetic strain,in which hyperglycemia occurs at 9 months of age. Thickenedcapillary BM and acellular capillaries in the retina have beenreported in this model (Miyamura and Amemiya, 1998; Matsuuraet al., 1999). Proliferative changes were reported in the pre-retinalvitreous of these rats, showing intra-retinal angiopathyaccompanied by newly formed vessels and significant hyalinizationof intra-retinal vessels (Tsuji et al., 2009). Hence, this might be usefulas an animal model for progressive DR, but the neovascularizationhas not been confirmed. Microaneurysms, the early clinical signof human NPDR, and arterio-venous shunts, which are associatedwith severe stages of human NPDR, were not observed in this model(Bhutto et al., 1999).

Otsuka Long-Evans Tokushima fatty ratsOtsuka Long-Evans Tokushima fatty (OLETF) rats spontaneouslydevelop type 2 diabetes with severe obesity. The retinalultrastructural changes observed in OLETF rats are similar tothose seen in diabetic individuals (Miyamura et al., 1999; Lu etal., 2003), but do not include hemorrhages or exudates. There issignificant thinning of the inner nuclear and photoreceptor layersof the retina, a decrease in the height of the retinal pigmentepithelial (RPE) cells, thickened capillary BM and poorlydeveloped basal infoldings in these rats. Abnormal ERG was alsoreported in sucrose-fed OLETF rats (Hotta et al., 1997). However,it has been suggested that OLETF rats are not suitable forstudying DR because the formation of acellular capillaries andpericyte ghosts typical of human DR are not accelerated in theserats (Matsuura et al., 2005).

Goto-Kakizaki ratsThe Goto-Kakizaki (GK) rat is a spontaneous model of non-obesetype 2 diabetes and develops chronic hyperglycemia at 4-6 weeksof age (Goto et al., 1988). Diabetic GK rats demonstrated anincreased ratio of retinal endothelial cells to pericytes (Agardh etal., 1997), and reduced retinal blood flow without changes in majorretinal vessel diameters, at an early stage of diabetes (Miyamoto etal., 1996). Because of the moderate and stable diabetic state, thisrat model is useful for investigating the retinal microcirculatorychanges caused by type 2 diabetes over an extended period of time(Miyamoto et al., 1996).

Spontaneously diabetic Torii ratsThe spontaneously diabetic Torii (SDT) rat is a non-obese type 2diabetes model that develops hyperglycemia at 20 weeks of age andcan survive for long periods without insulin treatment. SDT ratsexhibit tractional retinal detachment with fibrous proliferation, andpossibly neovascularization, without retinal ischemia. Thedevelopment of neovascularization in the absence of ischemiamakes this model considerably different from theneovascularization that has been observed in diabetic individuals(Yamada et al., 2005). Thus, the model needs additional study beforeit can be considered as a model of DR. Studies showed a reductionin the amplitude of ERG b-waves and OPs. Because OPs arisingfrom amacrine cells are a sensitive measure of retinal ischemia,these studies indicate the development of inner retinal ischemia inthese rats (Sasase, 2010).

Biobreeding ratsThe biobreeding (BB) rat is a spontaneous model of type 1 diabetesthat develops diabetes between the age of 40 and 140 days. Theserats exhibit retinal lesions, including pericyte loss, BM thickening,capillary degeneration and an absence of microaneurysms after 8-11 months of diabetes (Sima et al., 1985). Pancreas transplantationhas been shown to inhibit the development of retinal microvascularlesions in this model (Chakrabarti et al., 1987). However, very fewstudies of DR have been reported using this model, so its advantagesand disadvantages cannot be judged.

Spontaneously diabetic mouse modelsNon-obese diabetic miceNon-obese diabetic (NOD) mice spontaneously develop type 1diabetes owing to autoimmune destruction of insulin-producingpancreatic -cells by CD4+ and CD8+ T cells (Makino et al., 1980).Studies have shown the loss of retinal microvessels, reducedperfusion of the retina and disordered focal proliferation of vesselsin NOD mice (Shaw et al., 2006; Lee and Harris, 2008). It is reportedthat angiotensin II and thromboxane mediates the venule-dependent arteriolar vasoconstriction. Only a few reports on DRhave been published using this model.

db/db miceThe db/db (Leprdb) mouse is deficient for the leptin receptor andspontaneously develops type 2 diabetes associated with obesity at4-8 weeks of age. Six-month-old db/db mice have been shown toexhibit early features of DR, such as pericyte and endothelial cellloss (Midena et al., 1989), BM thickening (Clements et al., 1998)and increased blood flow (Tadayoni et al., 2003) in the retina. By

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15 months, these mice demonstrated distinct DR symptoms,including blood-retinal barrier (BRB) breakdown, pericyte loss,neuroretinal apoptosis, glial reactivation, possibleneovascularization and acellular capillaries in the retina (Cheunget al., 2005). Reports show that db/db mice have some specificadvantages for the study of the retinal microcirculation. These miceare darkly pigmented and hence the fluorescence of labeledelements circulating in the choroid can be masked by their pigmentepithelium.

Ins2Akita miceThe Ins2Akita (Akita) mouse contains a dominant point mutationin the gene encoding insulin-2 that induces spontaneous type 1diabetes in the B6 mouse strain. Heterozygous male Akita micedevelop hyperglycemia as early as 4 weeks of age. After 12 weeksof hyperglycemia, the retinas were found to have increased vascularpermeability, degenerate capillaries and alterations in themorphology of astrocytes and microglia with increasing durationof diabetes. Furthermore, increased retinal cell apoptosis wasidentified, accompanied by a distinct reduction in the thickness ofthe inner plexiform layer and inner nuclear layer (Barber et al.,2005). These mice showed loss of RGCs from the peripheral retinawithin the first 3 months of diabetes, as well as marked alterationsto the morphology of surviving cells (Gastinger et al., 2008).Another study demonstrated a significant reduction in the totalnumber of cholinergic and dopaminergic amacrine cells in Akitamice (Gastinger et al., 2006). This model has been studied up to15-18 months of age, but mortality increased significantly towardsthe end of this duration (T. S. K., unpublished data).

This model could be useful for exploring the molecularmechanisms involved in the initiation and early progression of DR.In addition, it is an ideal model for evaluating the neuroprotectiveeffects of drugs because of the quantifiable loss of RGCs in arelatively short time (4-5 months). Use of this model is increasingamong researchers interested in DR.

Mouse models of proliferative retinopathyDespite many attempts to establish suitable models that accuratelyreflect the features of late human DR, very few animal modelsdevelop severe DR with large areas of retinal non-perfusion andneovascularization. Hence, researchers have turned to non-diabeticanimals to study proliferative retinopathy. The widely used rodentmodels of proliferative retinopathy to study neovascularization arethose in which VEGF is overexpressed in photoreceptors (such asKimba mice and mice in which VEGF expression is driven by therhodopsin promoter) (Okamoto et al., 1997; Tee et al., 2008), miceoverexpressing insulin-like growth factor-1 (IGF1) in the retina(Ruberte et al., 2004), oxygen-induced retinopathy (Gole et al., 1990;Holmes and Duffner, 1996) and branch retinal vein occlusion(Zhang et al., 2007). These models induce possibleneovascularization and retinal detachment in the absence ofdiabetes. For example, transgenic mice with increased expressionof IGF1 in the retina exhibit signs of diabetes-like eye conditions,including pericyte loss, retinal capillary BM thickening, venuledilatation, IRMAs and possibly retinal neovascularization (Ruberteet al., 2004). Kimba mice, generated through photoreceptor-specificoverexpression of human VEGF165 protein, demonstrate retinalneovascular changes, increased permeability, pericyte and

endothelial cell loss, vessel tortuosity, leukostasis, and capillaryblockage, dropout and hemorrhage (Tee et al., 2008).

Akimba miceA potential transgenic mouse model of DR named ‘Akimba’ hasbeen developed recently by crossing Kimba mice with Akita mice(Rakoczy et al., 2010). These mice showed key features exhibitedby the parent strains: overexpression of VEGF (as in Kimba mice)and spontaneous type 1 diabetes (as in Akita mice). In this model,advanced retinal lesions resembling human PDR were caused indiabetic mice by ‘on top’ alternative approaches (e.g.neovascularization due to transgenic expression of humanVEGF165 in photoreceptors). Interestingly, vascular changes in thismodel include microaneurysms, increased prevalence of leakycapillaries, venous beading, tortuous vessels, capillary dropout andattenuation of vessels. Fig. 5 shows a comparison of retinal fundus,FFA and histology of Kimba, Akita and Akimba mice.

The neovascular changes observed in the Akimba mouse are notdue to long-term hyperglycemia, as in human DR, but are due tothe presence of the human VEGF165 transgene in thephotoreceptors. Hence, this model might not be suitable forstudying the etiology of DR or the factors associated with thedevelopment of pre-retinal neovascularization. The mechanismsof enhanced vascular and neuronal retinal changes,neuroprotection and the role of inflammation in Akimba mice havenot yet been studied. This newly developed model is an importanttool to improve our understanding of the complex processesunderlying the progression of DR.

Galactosemia modelsGalactosemia animal models of DR are induced in rodents bysupplementing with a diet containing 30-50% galactose. Galactose-fed rats and mice develop retinal microangiopathy that resemblesthe early stages of DR (Kern and Engerman, 1995), includingpericyte loss, acellular capillaries, thickened retinal capillary BM,IRMAs and, in dogs, also microaneurysms and intra-retinalhemorrhages. Galactosemic mice showed increased retinal capillarywidth and microaneurysms at 8 months of age. Retinalmicroaneurysms, acellular capillaries, pericyte ghosts and capillaryBM thickening became increasingly prevalent in mice on the 30%galactose diet for longer durations. The development of cataractshas been extensively probed using galactosemic rats and mice (Kernand Engerman, 1996a). Galactosemic rodents lack many of themetabolic abnormalities that are characteristic of diabetes, butdevelop many of the retinal complications of diabetes and thus canbe considered a valuable tool for the study of the pathogenesis ofdiabetic complications.

Large animal models of DRDiabetic dogsRetinopathy that develops in spontaneously or experimentallyinduced diabetic dogs is morphologically similar to human DR(Kern and Mohr, 2007; Kern, 2009; Zheng and Kern, 2010). Studiesof retinopathy using dog models have used mostly type 1 diabetesinduced by alloxan, STZ, growth hormone or pancreatectomy.Studies have also been carried out on long-term galactose-fed dogs(Engerman and Kern, 1984). Retinal lesions reported in diabeticdogs include microaneurysms, degenerate (acellular and non-

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perfused) capillaries, pericyte loss, IRMAs, thicker capillary BM,and dot and blot hemorrhages (Gardiner et al., 1994; Kern andEngerman, 1996b). Similar to diabetic dogs, long-term galactose-fed galactosemic dogs have retinal lesions that resemble those seenin human DR (Takahashi et al., 1993; Kern and Engerman, 1996b;Kobayashi et al., 1998). Galactose-fed dogs have been shown todevelop diabetes-like retinal vessel changes associated with boththe early and moderately advanced stages of retinopathy. However,it is 3-5 years before severe retinopathy develops, accompanied byoccasional intra-retinal neovascularization, in dog models(Engerman and Kramer, 1982; Engerman and Kern, 1984; Takahashiet al., 1992; Wallow and Engerman, 1997). In addition, the cost,lack of specific antibodies or molecular reagents, heightened ethicalconcerns and difficulty in maintenance mean that dog models areless useful than small animal models for the study of DR (Kobayashiet al., 1998).

Diabetic catsMost studies of diabetic cats involve type 1 diabetes induced bySTZ and pancreatectomy, with or without alloxan. Retinal lesionsinclude capillary BM thickening, increased vessel tortuosity,capillary non-perfusion, microaneurysms, fluorescein leakage andpossibly neovascularization (Mansour et al., 1990; Hatchell et al.,1995; Linsenmeier et al., 1998; Budzynski et al., 2005). Diabetic cats

develop only mild cataract, which allows the use of FFA and otherin vivo measurements for years after the induction of diabetes(Salgado et al., 2000; Richter et al., 2002). Cats also exhibit retinalhypoxia in diabetes, which might result from capillary plugging oraltered flow through microaneurysms (Linsenmeier et al., 1998).However, the cost, lack of specific antibodies or molecular biologyreagents, and slow development of lesions has made cat modelsless suitable than rodent models for studies of DR.

Diabetic pigsThe structure of the retinal vascular system in pigs is very similarto that of humans, which makes them very useful for research oneye diseases. Pigs developed retinal capillary BM thickening withseveral ultrastructural features, such as lamellation and rarefactionwithin BM, as early as 18 weeks after STZ treatment (Lee et al.,2010). Another study of type 1 diabetic pigs reported the fairly rapiddevelopment of features characteristic of early retinalmicrovasculature changes (Hainsworth et al., 2002). Further studiesusing this model might improve our understanding of DRprogression, and might provide an important platform forinvestigating new treatments that prove promising in small animalstudies, before progressing to clinical trials in humans. Thedisadvantages of pig models include high cost, lack of specificmolecular reagents and antibodies, difficulty in maintenance, and

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Fig. 5. Comparison of retinal fundus, FFA and histology of Kimba, Akimba and Akita mice. Fundus, FFA and retinal histology of Kimba (A-C), Akimba (D-F)and Akita (G-I) mice (courtesy of Lions Eye Institute, Perth, Australia). FFA shows the differences in retinal vasculature between Kimba (B), Akimba (E) and Akita (H)mice. Kimba (B) and Akimba (E) mice have foci of fluorescein leakages (arrows). Akita (H) mice have sharply defined retinal vessels and retinal capillary networkwith vessels radiating from the optic nerve head. Light micrographs of paraffin-embedded eyes of Kimba (C), Akimba (F) and Akita (I) mouse eyes show retinalhistology. The sections were stained with H&E. Arrows in C and F point to breaks in the RPE cells of Kimba and Akimba mouse eyes, whereas arrows in (I) point tointact RPE cells in Akita eyes. Ganglion cell layer (GCL); inner nuclear layer (INL); inner plexiform layer (IPL); outer plexiform layer (OPL); and outer nuclear layer(ONL).

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the fact that the techniques for genetic characterization andmanipulation are currently less precise than in small animalmodels.

Diabetic non-human primatesThe structural similarity of primate eyes to human eyes makes thempotential models for research on eye diseases. The main advantageof primate models over the other models discussed above is thepresence of the macula, an important site of damage in DR. Themost common primate models used for studies of DR includerhesus monkeys with diabetes induced by alloxan, STZ or totalpancreatectomy. Cynomolgus monkeys (Macaca fascicularis) andobese rhesus monkeys (Macaca mulatta) that develop diabetesspontaneously have also been used. Evidence shows thatretinopathy develops very slowly in diabetic non-human primates(as in humans). STZ- or pancreatectomy-induced diabetic monkeysstudied for up to 15 years showed retinal ischemia and defects inthe BRB and macula. However, these models lack vascular lesionsobserved in human DR (Tso et al., 1988). Studies of aged monkeyswith spontaneous diabetes revealed IRMAs and macular edema.Microaneurysms were also associated with the areas of non-perfusion (Johnson et al., 2005). Investigations on type 2 diabeticprimate models revealed hemorrhages, large areas of retinalcapillary non-perfusion, microaneurysms, cotton wool spots, intra-retinal hemorrhages and hard exudates in the macula (Kim et al.,2004). To date, retinal neovascularization or neuronal degenerationhas not been reported in diabetic non-human primates.

Despite the structural similarity of the eye in humans and non-human primates, these models are not always preferred over otheranimal models owing to several limitations. Non-human primatesare difficult to manipulate genetically, and molecular reagents forexperimental studies are lacking. They also have a longer gestationalperiod and lower birth rates. Other disadvantages include slowprogression of DR, increased maintenance costs and the ethicalissues of using non-human primates as animal models.

Future directionsAs outlined in this Perspective, animal models of DR are importanttools that will continue to enhance our understanding of thepathogenesis of DR, as well as the development of novel therapeuticapproaches. However, it is clear that there is still no perfect animalmodel that recapitulates all aspects of human DR. Although mostof the models discussed have demonstrated the basic features ofNPDR, the key feature of human PDR – pre-retinalneovascularization secondary to diabetes per se – is notrecapitulated in diabetic animal models. Nevertheless, currentclinical tools to treat existing PDR are relatively successful, and thusthe focus of future research should be to inhibit development ofthe early stages of the retinopathy, thereby preventing thesubsequent progression to advanced retinopathy. Notably, retinalthickening (consistent with DME) has been detected in a smallnumber of available models such as monkeys. Vision loss orimpairment is also now being studied in mice and rats using theoptokinetic response (Thomas et al., 2010); investigating the causeof a diabetes-induced reduction in visual function might providenew insight into the causes of vision loss in diabetic humans.

Some investigators feel that a remaining challenge is to developanimal models that mimic the progression of DR from the non-

proliferative to proliferative stage, as in human DR (Rakoczy et al.,2010; Li et al., 2010). This might be accomplished by acceleratingthe retinopathy in models through superimposing a particulargenetic or metabolic abnormality on top of diabetes (e.g. as ineNOS–/– or Akimba mice). Nevertheless, the extent to which theunderlying pathogenesis of retinopathy in such models mimics thatof human DR will be an important issue to address.

The application of appropriate techniques for thecharacterization of DR is an equally important challenge for thefuture. The current methods for in vivo characterization of DR,including ERG, FP, FFA and OCT, can monitor progressivepathological changes (vascular and non-vascular) in the sameanimal over time, but new methods that provide additionalinformation (e.g. retinal vascular caliber measurements) are needed.

In terms of clinical issues, many areas of uncertainty remain. Itis well known that, despite controlling systemic risk factors ofhyperglycemia and hypertension, many patients still progress todevelop the vision-threatening stages of DR (either PDR or DME).The current standard of care is laser therapy, which is inherentlydestructive, associated with side effects and is ineffective inreversing visual loss. New approaches, including intraocularadministration of anti-VEGF agents, are promising but havepotential risks (Cheung et al., 2010). Thus, preventing DR andtargeting early stages of DR is desirable. For example, the ability toprovide effective topical therapies that target multiple pathwaysunderlying retinal neovascularization and edema could furtherimprove the current management strategies for DR. Developmentof such therapies requires substantial basic and experimentalstudies, for which appropriate animal models of DR are essential.

ConclusionsNew and cost-effective therapies for treating DR, particularly theearly stages of DR, are urgently needed. Animal models thatdevelop lesions that are characteristic of human DR will continueto play a crucial role in understanding pathogenesis and for testingnew therapies before clinical trials. The success of each studydepends largely on the choice of the appropriate animal model,which, in turn, is driven by experimental design and focus. Keyfeatures to consider when choosing an animal model of DR include:the structural and biochemical features of the visual systemcompared with humans; the ability to perform geneticmanipulations; the availability and cost of the model; methodsavailable for disease characterization and validation; the timecourse of pathological changes; and ethical, moral and legal issues.Overcoming current challenges in DR research requires moreextensive experiments with the most promising models,incorporating advanced techniques for more accurate phenotyping.This approach will ultimately help to identify the best systems tobetter understand, prevent and treat the human disease.COMPETING INTERESTSThe authors declare that they do not have any competing or financial interests.

FUNDINGThis work was funded by the National Medical Research Council, ExploratoryDevelopmental Grant [NMRC/EDG/R797/53/2011 and NMRC/SERI-Retina Start-up/R603/37/2008 to V.A.B.]; Biomedical Research Council, Translational ClinicalResearch (TCR) Partnership [TCRP0101672B/R826/2011 to T.Y.W.]; and NationalMedical Research Council, New Investigator Grant [R751/35/2010 to S.S.C.].

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update on animal models of diabetic retinopathy: from ...increased flux through the polyol pathway,...

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